Optimisation of use of waste materials by Rhodopseudomonas palustris
A major challenge facing humanity is to combat anthropogenic climate change, and decrease our dependence on fossil fuels. One approach that has gained increasing popularity over the last few decades has been the generation of power by biodiesel, derived from plants, rather than traditional fossil fuels. However, a consequence of this increase in biodiesel manufacture is an increase in the production of waste glycerol – a low-value by-product. There is currently no cost-effective, ecologically sound solution for how to use or dispose of this waste glycerol. However, metabolising that waste biologically (bioremediation) is an option with considerable potential, and *Rhodopseudomonas palustris* is a strong candidate for bioremediation of waste glycerol. *Rhodopseudomonas palustris* is a hardy, toxin-resistant, and metabolically flexible bacterium. These traits make it an ideal candidate for breaking down waste glycerol, which is of varying composition, and may contain some toxic components. Although bioremediation of waste glycerol has previously been demonstrated to be possible using *R. palustris*, it is not currently economically feasible. It is therefore important to investigate ways to add value to waste remediation i.e. waste valorisation. This thesis investigates ways to increase the potential of *Rhodopseudomonas palustris* to valorise waste glycerol. In particular, this thesis addresses the possibility of using *R. palustris* for the production of cyanophycin, which would allow storage of carbon and nitrogen, and is potentially a high-value product. Cyanophycin production could allow for the simultaneous remediation of glycerol and urea (a nitrogenous waste from e.g. livestock manure slurries). This thesis first reports on the evolutionary history of an alternative cyanophycin producing enzyme, CphA2 (distinct from the widely studied CphA1). Phylogenetic analysis reveals for the first time that CphA2 is found outside of the cyanobacteria, and that there have been several horizontal gene transfers of the *cphA2* gene. An engineered strain of *R. palustris* carrying a heterologous *cphA1* gene is generated. The strain is shown to be able to synthesise cyanophycin. The transcriptome of this strain is analysed by RNAseq, and shown to be mostly unaffected by the expression of *cphA1*, except for unexpected increases in some transcripts associated with the degradation of aromatic compounds. The thesis then considers the development of new genetic tools for *R. palustris*. It tests antibiotic and counter-selection markers. It finds that chloramphenicol with its associated resistance gene is a useful selection marker, but that spectinomycin and its resistance gene are less effective. A combination of promoter and ribosome binding site is reported that directs a high level of gene expression; this is a useful new tool for future genetic engineering attempts. Finally, the metabolism of glycerol as a carbon source is investigated using flux analysis, showing that glycerol metabolism utilises the TCA cycle and glyoxylate cycle very little, and that a large amount of carbon fixation and PEP carboxylation occur. Additionally, possible ways to improve flux for cyanophycin synthesis by metabolic manipulation are considered.
biochemistry, genetic engineering, microbiology, molecular biology, purple bacteria, waste, waste remediation
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